The development of HIV virus strains that are resistant to existing protease inhibitors is one of the major obstacles to successful long-term antiretroviral therapy. We are investigating the structural changes of HIV-1 proteases from naturally occurring clinical variants that have become resistant to licensed protease inhibitors. The long-term goal of this project is to study the structural basis of HIV protease drug resistance and to use that knowledge to devise strategies to overcome resistance. The hypothesis being tested is that HIV-1 protease multidrug resistance is associated with mutations that expand the active site cavity of the protease, and as a result the inhibitors bind with lower affinity to the variant forms of the enzyme. The short-term goal of the project is to perform structure-function studies with a set of four multidrug-resistant HIV-1 protease clinical isolates. To accomplish this we will: Aim 1. Define the structural basis for multi-drug resistance in MDR HIV-1 protease variants and perform functional analyses of MDR HIV-1 protease variants to develop three dimensional structure-activity relationships that explain, in terms of protein-ligand interactions, why the drugs inhibit these structures less well compared to the wild-type. Aim 2. Design, synthesize and test small molecule inhibitors against the MDR HIV-1 protease variants. The HIV-1 protease variants described in Aim 1 will be subjected to X-ray crystallographic and functional studies. Structural studies are focused at discovering the three-dimensional changes in the protease variants. Mutant protease-ligand structures will be compared to wild-type protease-ligand structures. Functional studies of the ligands to the altered protease variants will be performed using surface plasmon resonance (BIACORE), isothermal titration calorimetry (ITC), enzyme assays and virologic drug susceptibility assays. Small molecule ligands that represent lead compounds against the MDR HIV-1 protease will be identified, synthesized and tested for efficacy. Peptide derivatives and peptidomimetic ligands will be constructed in four categories: modified peptides, retro-inverso peptides, peptoids and amide isosteres. Solving the structural problem of why certain mutant HIV-1 proteases are not inhibited by the standard inhibitors and using this information to design new and effective compounds are of a great importance to human welfare.